Project Summary/Abstract Cardiovascular disease is responsible for approximately 17 million global deaths each year and is therefore a critical issue in worldwide human health. One area of cardiovascular biology that is currently in pressing need of new therapies is the repair of myocardial infarction (MI), which afflicts one American every 34 seconds. Following MI, billions of myocytes are typically lost within the first days. These myocytes are not regenerated but are instead replaced by fibrosis. Replacement of necrotic cardiomyocytes with exogenous cardiomyocytes derived from pluripotent stem cells has been shown to improve heart function after infarction in pre-clinical animal models. However, the critical challenge of creating large cardiac grafts that are well integrated with the host tissue has plagued the cell-based repair field since its inception, creating serious safety and efficacy concerns. For example, poor graft cell seeding efficiency and cell survival limit the efficacy of this approach. Additionally, graft cardiomyocytes are often separated from host cardiomyocytes by infarct scar tissue, which may result in arrhythmias caused by dyssynchronous coupling between graft and host. Finally, vascular networks connecting the host tissue to the graft are often dispersed and unorganized, limiting the survival and metabolic potential of grafted cells. To address these issues, I will build an integrated engineered human cardiac tissue platform, which can be remotely activated to degrade the local infarct scar as the tissue becomes vascularized. I will first develop a `thermogenetic' system that enables remote transient activation of protease overexpression only at the graft site and in concert with graft expansion after implantation. This controlled approach is necessary because ubiquitous overexpression or delivery of proteases could cause adverse myocardial remodeling or rupture. For improved vascularization, I will use a microtissue molding approach to test whether the presence of highly aligned microvascular `cords' in scaffold-free cardiac tissue guides microvascularization and accelerates tissue perfusion. I will then test whether together these systems enable improved engraftment, perfusion, and heart function following MI. This proposal is based on my previous experience in constructing and grafting scaffold-free human heart tissue from pluripotent stem cells, controlling grafted cells using gene therapy, and using microfabrication techniques to spatially organize multicellular tissue architecture. This work will represent the first steps towards comprehensive remote activation and control of engineered tissues after engraftment in vivo, introducing a new paradigm for precision post-implantation control of cell-based therapies for regenerative medicine applications.